Light regulates the degradation of the regulatory protein VE-1 in the fungus Neurospora crassa

Gil‑Sánchezetal. BMC Biology (2022) 20:149 https://doi.org/10.1186/s12915‑022‑01351‑x RESEARCH ARTICLE Light regulates thedegradation oftheregulatory protein VE‑1 inthefungus Neurospora crassa María del Mar Gil‑Sánchez, Sara Cea‑Sánchez, Eva M. Luque, David Cánovas and Luis M. Corrochano* Abstract Background: Fungi use light as an environmental signal to regulate developmental transitions that are key aspects of their biological cycles and that are also relevant for their dispersal and infectivity as plant or animal pathogens. In addition, light regulates the accumulation of photoprotective pigments, like carotenoids, and other secondary metabolites. Most fungal light responses occur after changes in gene transcription and we describe here a novel effect of light in the regulation of degradation of VE‑1, a key component of the velvet complex, in the model fungus Neurospora crassa. The velvet complex is a fungal‑specific protein complex that coordinates fungal development, secondary metabolism, and light regulation by interacting with other regulators and photoreceptors and modifying gene expression. Results: We have characterized the role of VE‑1 during conidiation in N. crassa. In vegetative mycelia, VE‑1 is localized in the cytoplasm and nuclei and is required for light‑dependent transcription but does not interact with the pho‑ toreceptor and transcription factor WC‑1. VE‑1 is more stable in light than in darkness during asexual development (conidiation). We have shown that this light effect requires the blue‑light photoreceptor WC‑1. We have characterized the role of the proteasome, the COP9 signalosome (CSN), and the adaptor component of cullin‑RING ubiquitin ligases, FWD‑1, in the degradation of VE‑1. Conclusions: We propose that this new effect of light allows the fungal cell to adapt quickly to changes in light exposure by promoting the accumulation of VE‑1 for the regulation of genes that participate in the biosynthesis of photoprotective pigments. Keywords: Velvet complex, Light regulation, Protein degradation, COP9 signalosome © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Developmental transitions in fungi are key to their success as plant and human pathogens. The development of appresoria by Magnaporthe oryzae and related fungi facilitates invasion of plant cells [1]. The production of conidia, spores, or ascospores facilitates dispersal and the colonization of new hosts by Aspergillus fumigatus and other pathogenic ascomycetes [2]. The development of resistance structures, sclerotia, facilitates later invasion of plants by Botrytis cinerea when environmental conditions are more appropriate [3]. Fungal development is regulated by environmental and internal signals. Asexual reproduction in Neurospora crassa, conidiation, is controlled by a circadian clock resulting in bands of conidia appearing approximately every 24h [4]. Exposure to air, desiccation, lack of nutrients, and light regulate asexual and sexual development in many fungi [5–8]. These environmental signals are perceived and signaled into the cell by using signal transduction pathways that often include Open Access *Correspondence: [email protected] Departamento de Genética, Universidad de Sevilla, Reina Mercedes s/n, 41012 Seville, Spain Page 2 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 heterotrimeric G-proteins and their receptors [9–11], and photoreceptors [12, 13]. In N. crassa, light is perceived through a blue-light photoreceptor complex, the White Collar Complex (WCC), a photoreceptor, and transcription factor complex composed of the photoreceptor and Zn-finger protein WC-1 and the Zn-finger protein WC-2. The WCC transiently binds to promoters to activate transcription [14, 15]. WC proteins have been described in most fungi together with other photoreceptors like phytochromes, cryptochromes, and rhodopsins [12, 13]. In Aspergillus nidulans, WC proteins interact with the phytochrome in a light sensing complex capable of sensing red light (phytochrome) and blue light (WC proteins) [16, 17]. The regulation of protein turnover plays a key role in fungal development. Early studies uncovered the role of the COP9 signalosome (CSN) in fruiting body development during the sexual cycle of A. nidulans [18, 19]. Mutations in the genes encoding CSN subunits in N. crassa result in growth defects, alterations in conidation, and misregulation of the circadian clock [20]. The CSN is a deneddylase that removes the ubiquitin-like protein Nedd8 from modified proteins and is composed of eight subunits in A. nidulans and seven subunits in N. crassa, including the CSN-5/CsnE subunit responsible for the Nedd8 isopeptidase activity [21]. The deneddylase activity of the CSN is a key element in the regulation of the ubiquitin pathway of protein degradation by the proteasome. Proteins are targeted for degradation after labeling with ubiquitin units by cullin-RING ubiquitin ligases (CRLs) in the ubiquitin–proteasome pathway [22, 23]. In N. crassa, the degradation of FRQ, the key regulator of the circadian clock, is mediated by the F-box protein FWD-1, the adaptor component of a CRL that interacts with target proteins to promote their ubiquitylation and degradation by the proteasome [24]. FWD-1, together with SKP-1 and CUL-1, form a type of CRL named SCF (SKP Cullin F-box) that mediates FRQ ubiquitylation and degradation [24, 25]. The activity of CRLs, including SCFFWD−1, is regulated by removal of the ubiquitinlike protein Nedd8 by the CSN. Active CRLs require the deneddylation activity of the CSN since it promotes the recycling of CRL components for interaction with additional target proteins. In addition, deneddylation of CRLs by the CSN prevents the degradation of the CRLs by excessive autoubiquitylation [26–29]. Mutations that render the CSN inactive result in reduced amounts of FWD-1, and increased protein stability, as observed by the increased stability of FRQ in N. crassa strains with mutations in genes encoding CSN subunits [20, 25]. Fungi can produce many secondary metabolites, among them penicillin and other antibiotics, as well as enzymes and toxic chemicals such as the carcinogenic aflatoxin that help them thrive and facilitate their pathogenic lifestyles [30]. Other secondary metabolites, carotenoids and melanins, protect fungi from oxidative stress and UV radiation [31, 32]. Fungal development and secondary metabolism are often coordinated by environmental signals such as light [33]. A key element in the coordination of fungal development and secondary metabolism is the velvet complex [33, 34]. This fungal-specific protein complex was first identified in A. nidulans and is composed of the velvet proteins VeA and VelB, and the methyltransferase LaeA [35]. The velvet proteins VE-1 and VE-2, and a LaeA homolog, LAE-1, have been identified and characterized in N. crassa [36, 37]. Velvet proteins are defined by the velvet domain, a 150-amino acid domain involved in dimer formation with a structure that resembles the DNA-binding fold of the mammalian transcription factor NF-κB [38]. The velvet complex is proposed to regulate transcription by DNA binding to promoter regions and chromatin modification [39]. The activity of the velvet complex is regulated by the subcellular localization of its components, and by interactions with other regulatory proteins. In A. nidulans, VeA enters the nucleus as a heterodimer with VelB and interacts in the nucleus with LaeA to form the trimeric velvet complex [35]. Entry of VeA/VelB to the nucleus requires the importin KapA and is regulated by light. VeA is located in the nuclei when the fungus is grown in the dark, but light promotes the accumulation of VeA in the cytoplasm [40]. VeA interacts with kinases and methyltransferases that regulate its phosphorylation and the subcellular distribution of the protein [39, 41]. In addition, VeA interacts with the red-light photoreceptor phytochrome (FphA), presumably to coordinate fungal development and light regulation [16]. VeA, thus, is a molecular scaffold in several protein complexes that allows the integration of environmental signals to modulate gene transcription and development [42]. Protein complexes composed of velvet proteins regulate gene expression in other fungi. In the human pathogen Histoplasma capsulatum, two velvet proteins interact with the transcription factor Ryp1 to regulate the transcription of genes related to virulence and cell morphology [43]. In N. crassa, the VeA homolog, VE-1, interacts with VE-2 and LAE-1 to form a velvet complex in vegetative hyphae where it regulates secondary metabolism, and the accumulation of carotenoid pigments. In addition, the N. crassa velvet complex participates in the regulation of sexual development and the formation of aerial hyphae, the initial stage of asexual development (conidiation) [36]. Despite the regulatory role of the velvet complex in the coordination of environmental sensing, secondary metabolism, and development, there is little information regarding the presence and subcellular localization of the Page 3 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 velvet complex during fungal development and how it regulates transcription. Here, we characterize the role of VE-1 in the transcriptional control of secondary metabolism using carotenoid biosynthesis as a model, and we show that its action is independent of the blue-light photoreceptor and transcription factor WC-1. In addition, we show that a novel light-controlled degradation process regulates the presence of VE-1 in aerial hyphae during conidiation in N. crassa. The regulation by light of VE-1 degradation is a new mechanism that modulates the amount of VE-1 and its availability to form the regulatory velvet complex. Our results underscore the coordination between light and developmental stage in regulating the availability of VE-1 for the formation of the velvet complex to regulate asexual development in N. crassa. Results VE‑1 isrequired fortheaccumulation ofcarotenoids andlight‑dependent transcription The velvet complex regulates the accumulation of the orange carotenoid neurosporaxanthin in vegetative mycelia of N. crassa as shown by the reduced accumulation of carotenoids after exposure to light [36, 37]. The decrease in carotenoid accumulation was reverted by transformation with a wild-type copy of ve-1 (Additional file1: Fig. S1a, b). In addition, VE-1 regulated the accumulation of carotenoids in conidia (Additional file1: Fig. S2a, b). Light induces the accumulation of carotenoids in mycelia after the activation by the WCC of the genes encoding the biosynthetic enzymes (al-1, al-2, al-3, and cao-2) [44]. The role of the velvet complex in the regulation of carotenoid biosynthesis and the possible interaction with the WCC are not understood. VE-1 regulates the expression of 15–20% of all N. crassa genes in vegetative mycelia, and the ve-1 mutant had a small reduction in the accumulation of the mRNAs for al-2 (encoding the phytoene synthase) and cao-1 (encoding a carotenoid oxygenase) under standard growth conditions [36]. The absence of VE-1 led to a tenfold reduction in the capacity to perceive light by vegetative hyphae as the threshold for light-dependent accumulation of carotenoids increased to 100J/m2 in the Δve-1 mutant compared to the wildtype threshold of 10J/m2 (Fig.1a). To figure out whether the reduction in the capacity to perceive light in the Δve-1 mutant was caused by alterations in the amount or activity of the photoreceptor WC-1 expression levels of light-dependent genes were analyzed. Light activates transiently the genes for the biosynthetic enzymes, photoadaptation, and the light-dependent mRNA accumulation of these genes was reduced in the absence of VE-1 (Fig.1b). A reduction in light-dependent mRNA accumulation has been observed for other light-regulated genes in the Δve-1 mutant, indicating that this effect on transcription is not limited to carotenoid genes (Additional file1: Fig. S2c). Photoadaptation correlates with a transient light-dependent phosphorylation of WC-1 [45]. However, the loss of sensitivity to light and the reduced light-dependent transcription of carotenoid genes were not due to changes in the amount or light-dependent phosphorylation of the photoreceptor WC-1 (Fig.1c). Our results suggested, instead, a direct regulatory role of VE-1, and the velvet complex, on lightdependent transcription in N. crassa nuclei. In order to confirm the nuclear localization of VE-1, we created a strain with VE-1 tagged with the FLAG epitope for the detection of the protein using a FLAG-specific antibody (Additional file 1: Fig. S2a). The new allele (ve-1FLAG) was under the control of the ve-1 native promoter as it replaced the original gene and will be used as the wildtype strain in the rest of this work. The ve-1 gene was weakly activated by light in vegetative hyphae (Additional file1: Fig. S2d), and the tagged VE-1 protein was detected in similar amounts in mycelia kept in the dark or exposed to light in the wild-type strain or in strains with mutations in the genes for the blue-light photoreceptors WC-1 or VVD (Additional file1: Fig. S2e). We detected VE-1 in the cytoplasmic and nuclear fractions, and the subcellular distribution of VE-1 did not change with light in the wild-type strain (Fig.1d), or in strains with mutations in the genes for the blue-light photoreceptors (Additional file1: Fig. S2f). These results supported the proposal that light-dependent activation of transcription in N. crassa requires the WCC and the velvet complex. Light prevents thedegradation ofVE‑1 inaerial hyphae andallows theaccumulation ofVE‑1 inthenucleus VE-1 and the velvet complex participate in the regulation of asexual development [36]. The absence of ve-1 led to short aerial hyphae, an early stage of conidiation [46], and this defect was reverted by transformation with the wild-type ve-1 gene (Additional file 1: Fig. S1a, c). The reduction in the length of aerial hyphae in the Δve-1 mutant was observed only in cultures kept in light. The effect of light on aerial hyphae length required the photoreceptor WC-1, since we did not detect any difference in the length of aerial hyphae of the Δve-1 Δwc-1 strain kept in dark or light (Fig.2a,b). The absence of ve-1 led to a reduction in the accumulation of conidia compared to the wild type, in particular when the Δve-1 strain was grown in light (Additional file1: Fig. S3), suggesting that the absence of VE-1 led to a light-dependent inhibition of conidiation by inhibiting the growth of aerial hyphae. We hypothesized that VE-1 should play its regulatory role in the nuclei of aerial hyphae and we used the strain with the ve-1FLAG Page 4 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 allele to monitor the abundance and subcellular localization of VE-1 during conidiation. For these experiments, we first incubated conidia from each strain in liquid media to promote vegetative growth for 24h. We then transferred the hyphae from liquid media to the air-exposed surface of a petri dish with solid minimal agar to induce conidiation and covered the vegetative mycelia with filter paper. After incubation in dark or light, we collected the aerial hyphae that grew through the filter paper and the supportive vegetative mycelia under the filter paper for RNA or protein extraction, and characterization [47] (Additional file1: Fig. S4a). We detected VE-1 in vegetative mycelia kept in dark or light, but we observed that light played a key role on the accumulation of VE-1 during conidiation. We could not detect VE-1 in aerial hyphae that had grown in the dark, but VE-1 was clearly observed in samples of aerial hyphae that had been exposed to light (Fig. 2c). The absence of VE-1 in aerial hyphae kept in the dark was surprising since we detected similar amounts of ve-1 mRNA in Fig. 1 VE‑1 regulates light‑dependent transcription and the accumulation of carotenoids. a Effect of light intensity on the accumulation of carotenoids. Mycelia of the wild‑type and Δve-1 mutant strains were grown for 2 days in the dark at 22 °C and illuminated for 2 min prior to incubation for 24 h in the dark at 8 °C. The plot shows the average and standard error for six measurements for each light intensity. b Light‑dependent transcription and photoadaptation. Wild‑type and Δve-1 mutant strains were grown for 2 days at 22 °C in the dark and then exposed to light during the times indicated prior to RNA purification and quantification by RT‑PCR. The plots show the average and standard error of the mean of the relative mRNA accumulation in three independent experiments. The results from each PCR for each gene were normalized to the corresponding PCR for tub-2 to correct for sampling errors. Then, the results were normalized to those obtained with the wild type after exposure to 30 min of light. c Light‑dependent phosphorylation of the photoreceptor WC‑1. Mycelial samples of the wild‑type and the Δve-1 mutant strains were grown for 2 days at 22 °C in the dark and then exposed to light during the times indicated. Total protein extracts were separated by SDS‑PAGE, and hybridized with an antibody specific for WC‑1. Two hundred micrograms of proteins was loaded per lane. Additional bands are due to the transient light‑dependent WC‑1 phosphorylation. As loading control, we used a Coomassie staining of each protein sample. d Subcellular localization of VE‑1. Mycelial samples of the ve-1FLAG strain were grown for 2 days at 30 °C in the dark, light, or grown in the dark and exposed to light during 30 min. Total protein samples (T), or samples enriched in cytoplasmic (C) or nuclear (N) proteins were separated by SDS‑PAGE, and hybridized with antibodies specific for FLAG or histone H3. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample Page 5 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 mycelia and aerial hyphae in cultures that had been kept in dark or light (Additional file1: Fig. S4b). The presence of a PEST domain (a protein segment rich in proline, glutamic acid, serine, and threonine) in VE-1 [37] suggested that VE-1 could be subjected to degradation by the proteasome. We, thus, hypothesized that VE-1 could be degraded in aerial hyphae kept in the dark and that light perceived through the photoreceptor WC-1 could prevent the degradation of VE-1 in aerial hyphae. In order to confirm this hypothesis, we performed similar experiments in a Δwc-1 strain. This strain accumulated very little VE-1 in aerial hyphae in all conditions, confirming the key role of light in preventing VE-1 degradation in aerial hyphae (Fig.2c). VE-1 was detected in both cytoplasmic and nuclear fractions, but the amount of VE-1 in the nuclear fraction of light-exposed aerial hyphae increased when compared to mycelial samples (Fig.2d). Our results suggested that VE-1 was degraded in aerial hyphae in the dark and that the degradation of VE-1 was reduced by exposure to light perceived through WC-1. We hypothesized that a light-regulated degradation process controls the abundance of VE-1 in aerial hyphae of N. crassa allowing the accumulation of VE-1 in nuclei to regulate transcription during development. VE‑1 isdegraded bytheproteasome andlight regulates thedegradation The mechanism of light-regulated degradation of VE-1 was assayed by treating N. crassa vegetative mycelia with cycloheximide, an inhibitor of protein synthesis. We could not detect VE-1 after 2h in cycloheximide unlike a stable protein like the nucleolar protein homologous to yeast Nop1p (Additional file1: Fig. S5a). The half-life of VE-1 in cycloheximide was about 100min, but we observed that the degradation of VE-1 could be modulated by light resulting in different degradation kinetics (Fig.3a). Continuous exposure to light reduced the degradation of VE-1 as observed in particular after Fig. 2 Light regulates the accumulation of VE‑1 during conidiation. a VE‑1 regulates the growth of aerial hyphae. Aerial hyphae of the wild‑type, Δve-1, Δwc-1, and Δwc-1 Δve-1 strains after 3 days of growth in minimal agar at 30 °C in dark or light. The length of aerial hyphae growing up from vegetative mycelia in two representative cultures is indicated by arrows. b Length of aerial hyphae in each strain after growth at 30 °C in dark or light. Average and standard error of the mean in 3–5 independent experiments. c Accumulation of VE‑1 during conidiation. Upper part. Protein samples from cultures kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 15 h (M15) or 24 h (M24) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 15 h (A15) or 24 h (A24) of the induction of conidiation. We used the ve-1FLAG and the Δwc-1 ve-1FLAG strains. Bottom part. Proteins were separated by SDS‑PAGE, and hybridized with an antibody specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. d VE‑1 accumulates in the nuclear fraction during conidiation. Upper part. We collected samples of the ve-1FLAG strain grown in the light from vegetative mycelia (M), supporting vegetative mycelia (M15) or (M24), and aerial hyphae (A15) or (A24). Bottom part. Total protein samples (T), or samples enriched in cytoplasmic (C) or nuclear (N) proteins were separated by SDS‑PAGE, and hybridized with antibodies specific for FLAG or histone H3. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample Page 6 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 120 min in cycloheximide, while the degradation of VE-1 increased after 30min of light when compared to cultures kept in the dark (Fig.3a). The effect of light modulating VE-1 stability required the photoreceptor WC-1 since the kinetic of VE-1 degradation in the Δwc-1 strain did not change under any light regime (Fig. 3a). WC-1 is a very stable protein (Additional file1: Fig. S5b), and it does not interact with VE-1 in co-immunoprecipitation assays (Additional file1: Fig. S5c) likely ruling out any direct regulation of WC-1 on VE-1 stability. In order to characterize the degradation of the other components of the velvet complex, VE-2 and LAE-1, we used N. crassa strains carrying ve-2HA or lae-1FLAG alleles for detection of VE-2 or LAE-1 with antibodies specific for HA or FLAG, respectively [36]. Unlike VE-1, we observed the accumulation of VE-2 and LAE-1 after long incubations in cycloheximide (480min) suggesting that VE-1 was the only component of the velvet complex subjected to degradation when protein synthesis was blocked (Additional file1: Fig. S5d). Our results confirmed that light regulates the amount of VE-1 by controlling its degradation in vegetative mycelia, but the role of the photoreceptor WC-1 remains to be understood. Identifying the mechanism of VE-1 degradation will help to understand the interactions between light, protein degradation, and VE-1 activity. To confirm the role of the proteasome in the degradation of VE-1, we used thiolutin, an inhibitor of JAB1/ MPN/Mov34 (JAMM) metalloproteases, such as Rpn11, the deubiquitinating enzyme of the 19S proteasome lid, and Csn5, the deneddylase of the COP9 signalosome (CSN) that regulates cullin-RING ubiquitin ligases. Thiolutin inhibits the ubiquitin–proteasome system (UPS) and protein turnover leading to increased protein stability [48]. Incubation of N. crassa vegetative mycelia with cycloheximide and thiolutin resulted in increased stability of VE-1 when compared with samples incubated with cycloheximide only (Fig.3b). This result supported the proposal that VE-1 was degraded by the proteasome. The degradation ofVE‑1 requires theCOP9 signalosome andtheadaptor protein FWD‑1 We then examined how VE-1 is targeted for degradation by the proteasome. The key role of the CRL adaptor protein FWD-1 on FRQ stability, and the circadian clock regulation on conidiation prompted us to characterize the role of FWD-1 and the CSN on the stability of VE-1. We created strains with deletions in fwd-1, csn-1, and csn-5 that, in addition, carried the ve-1FLAG allele for detection of VE-1 in protein extracts by crossing mutant strains with the wild-type strain carrying the ve-1FLAG allele, and characterizing the progenies to identify strains with mutant and ve-1FLAG alleles. We selected strains with mutations in csn-5 and csn-1 as two representatives of the main subunits of the CSN. CSN-5 is the JAMM-domain subunit of the CSN responsible for Nedd8 cleavage from cullin proteins, and CSN-1 is one of the several Proteasome, COP9 signalosome, and eukaryotic initiation factor 3 (PCI)-domain proteins in the CSN [20, 49]. Vegetative mycelia from the three mutant strains were incubated with cycloheximide and the stability of VE-1 was assayed in protein extracts. VE-1 was degraded after incubation with cycloheximide in the Δcsn-1 strain following a kinetics and light regulation similar to that observed previously in the wild-type strain, but mutations in csn-5 or fwd-1 prevented the degradation of VE-1, even after incubations of 8h in cycloheximide (Fig.3c). It seems that the absence of CSN-1 did not impair the capacity of the CSN to regulate the degradation of VE-1, but the Nedd8 cleavage activity provided by CSN-5 was a key element for the role of the CSN in the regulation of VE-1 degradation. These mutations did not modify the lightdependent phosphorylation of WC-1 or the subcellular (See figure on next page.) Fig. 3 The degradation of VE‑1 by the proteasome is regulated by light. a Regulation by light of VE‑1 degradation. Cultures of vegetative mycelia of the ve-1FLAG and the Δwc-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were collected at different times. Proteins were separated by SDS‑PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Ponceau staining of each protein sample. Each hybridization was quantified using as reference the amount of VE‑1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of four independent experiments. b The proteasome inhibitor thiolutin prevents the degradation of VE‑1. Cultures of vegetative mycelia of the ve-1FLAG strain were grown at 30 °C in liquid media and exposed to 30 min of light, then cycloheximide and/ or thiolutin were added to the cultures, and samples were removed at different times. Cultures without any additional chemical added were used as controls. Proteins were separated by SDS‑PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. The hybridization was quantified using as reference the amount of VE‑1 detected at time point 0 (before addition of cycloheximide, thiolutin or both). The plot shows the average and standard error of three independent experiments. c FWD‑1 and the CSN participate in the degradation of VE‑1. Cultures of vegetative mycelia of the Δcsn-1 ve-1FLAG, Δcsn-5 ve-1FLAG, and Δfwd-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light, or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were removed at different times. Proteins were separated by SDS‑PAGE and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. Each hybridization was quantified using as reference the amount of VE‑1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of 2–3 independent experiments Page 7 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 Fig. 3 (See legend on previous page.) Page 8 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 distribution of VE-1 in vegetative mycelia (Additional file1: Fig. S6), only the stability of VE-1. Our results suggest that the degradation of VE-1 requires the SCFFWD−1 and its adequate regulation by the CSN. VE‑1 accumulates inaerial hyphae andconidia ofstrains withmutations ingenes oftheCOP9 signalosome ortheadaptor protein FWD‑1 The experiments that assayed the stability and degradation of VE-1 in the wild-type strain and mutants were carried out in vegetative mycelia incubated in liquid media with cycloheximide. Since we did not detect VE-1 in aerial hyphae kept in the dark, we hypothesized that SCFFWD−1 and the CSN had a key role in the degradation of VE-1 in aerial hyphae and that this degradation would be reduced by exposure to light. Thus, we expected an increased accumulation of VE-1 in aerial hyphae of strains with mutated csn subunit genes or fwd- 1. Mutants in subunits of the CSN or in fwd-1 show slow growth and delayed conidiation [20, 24, 25], presumably due to the major alteration in protein stability caused by these mutations. We grew the Δfwd-1 mutant mycelia for 48h to induce conidiation and to isolate sufficient aerial hyphae for protein extraction and detection, but its slow growth rate prevented direct comparisons with the wild-type strain. However, we could detect VE-1 in aerial hyphae of the Δfwd-1 mutant strain that had been incubated in the dark or light, unlike our previous observations with aerial hyphae of the wild-type strain (Figs.4a and 2c). In addition, we detected similar amounts of VE-1 in conidia isolated from dark- or light-grown Δfwd-1 or Δcsn-5 mutant strains (Fig.4b). As expected, the amount of VE-1 in conidia from the wild-type strain was higher in samples isolated from cultures kept in the light, than in the dark (Fig.4c). Our results supported the proposal that the reduction in the amount of VE-1 in aerial hyphae kept in the dark was due to its degradation by the proteasome in a process regulated by SCFFWD−1 and the CSN. The reduction ofVE‑1 degradation bylight allows rapid accumulation ofVE‑1 afterdark tolight transitions Our results show that VE-1 protein turnover is regulated by light. Therefore, the biological role of this light control was investigated. Light often activates fungal transcription producing the accumulation of light-dependent mRNAs and proteins [12, 13]. However, the accumulation of light-induced proteins, and their biological effect, are likely to occur several minutes after the onset of illumination. In N. crassa, maximum mRNA accumulation from early light-responsive genes is observed with exposures of 15–30min of light [44]. Short exposure times, for example 5s, activate transcription of the conidiation genes con-10 and con-6, but maximum mRNA accumulation was observed after 45min of further incubation in the dark [50]. It is possible that light-dependent transcription and translation is not sufficient as a response to sudden changes in light exposure or intensities in natural habitats, such as the surface of burned trees where conidiating mycelia of Neurospora species are often observed [51, 52]. We can hypothesize that the regulation by light of VE-1 degradation could be a novel mechanism that ensures the presence of VE-1 for further transcriptional regulation during the development of conidia after sudden exposures to light. We thus would expect quick changes in the accumulation of VE-1 in aerial hyphae after a transition from dark to light. In order to prove this hypothesis, we incubated aerial hyphae of the wild-type strain for 15h in the dark and then transferred the cultures to light for a variable time, from 5 to 120min, prior to sample isolation and protein extraction. As expected, Fig. 4 Light regulates the accumulation of VE‑1 in aerial hyphae by preventing its degradation. a The degradation of VE‑1 in aerial hyphae requires FWD‑1. Protein samples from cultures of the Δfwd-1 ve-1FLAG strain kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 48 h (M48) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 48 h (A48) of the induction of conidiation. Proteins were separated by SDS‑PAGE and hybridized with antibodies specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. b The degradation of VE‑1 in conidia requires FWD‑1 and an active CSN. Protein samples were isolated from conidia of the ve-1FLAG, Δcsn-5 ve-1FLAG, and Δfwd-1 ve-1FLAG strains isolated from cultures kept in the dark or light. Proteins were separated by SDS‑PAGE and hybridized with antibodies specific for FLAG. Fifty micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. c Light promotes the accumulation of VE‑1 during conidiation. Protein samples from cultures of the ve-1FLAG strain kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 15 h (M15) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 15 h (A15) of the induction of conidiation. For the dark to light transfer or light to dark transfer, the M15 or A15 samples were incubated in light or dark, respectively, for the indicated times after 15 h of induction of conidiation in dark or light, respectively, and collected for protein purification. Proteins were separated by SDS‑PAGE and hybridized with an antibody specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. d A model for the regulation of VE‑1 degradation during conidiation and light. VE‑1 accumulates in the cytoplasm and nuclei of vegetative hyphae following transcription and translation. VE‑1 is degraded by the proteasome in a process that requires interaction with the SCFFWD−1 and ubiquitylation (symbolized by stars) and is regulated by the CSN and light. During the development of aerial hyphae VE‑1 is highly degraded but light reduces VE‑1 degradation and promotes its translocation to the nucleus for gene regulation (See figure on next page.) Page 9 of 15 Gil‑Sánchezetal. BMC Biology (2022) 20:149 VE-1 was detected in vegetative mycelial samples but not in aerial hyphae after 15h of growth in the dark. However, 5min of further incubation in light was sufficient to prevent degradation of VE-1, allowing the detection of the protein in aerial hyphae (Fig.4c). The opposite experiment, the transfer of light-exposed aerial hyphae to the dark, always led to accumulation of VE-1, at least up to 120min of incubation in the dark (Fig.4c). Our results suggest that the light-dependent regulation of VE-1 degradation is a novel mechanism that ensures the presence of VE-1 to regulate transcription during the response to light in the aerial hyphae of N. crassa. Discussion The velvet complex coordinates fungal development, secondary metabolism, and light responses by regulating gene expression (33, 34). In N. crassa, the three components of the velvet complex (VE-1, VE-2, and LAE-1) interact in vegetative mycelia to regulate transcription [36], and we show here that the accumulation of VE-1 Fig. 4 (See legend on previous page.)